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#LabHacks: To compensate or not to compensate, that is the question

By Charleen Salesse

One of the most basic but divisive questions when learning patch-clamp recording techniques implicates the usefulness of series resistance compensation. From tutors to experimenters, from rookies to old and wise supervisors, answers and justifications fluctuate. Let’s take a quick look at that question and try to refresh the basic concept.

​ What is series resistance?

Series resistance is the sum of all resistances between the amplifier and the inside of the cell. It is mostly made up of the size and shape of the tip of the pipette and anything that blocks it, and the movement of ions. Series resistance limits the amount of current used to clamp the cell membrane.

The problem is that you are injecting current and recording electrical signals with the same electrode. This leads to a certain error in your measurements. In voltage clamp, to keep the membrane potential stable, the current injected should be equal to the electrical conductance of the cell membrane. It is less of a problem in current clamp as the current you are injecting is constant.

How is compensating done?

There are ways to reduce (not remove) this error, such as using low resistance pipettes and measuring smaller currents (the larger the conductance, and thus the current at a given holding potential, the larger the error). The error can also be predicted electronically, a technique known as series-resistance compensation. The predicted discrepancy can be used to reduce the error. To compensate, a current proportional to the difference of the measured membrane potential to the set one is injected.

The problem is that the error is not stable. In current clamp, the series resistance can easily be estimated, and compensating for voltage variations across the series resistance of the cell is achieved simply by a bridge balance circuit, which compensates for the linear error in voltage. All modern amplifiers and software now integrate such features easing the process for experimenters.

When should you compensate?

Uncompensated series resistance has major problems, such as voltage drops, which is the difference between the command and the membrane potential caused by the resistance; the temporal resolution error, which is the time it takes to get to the imposed potential by the cell; and a reduced bandwidth, which increases the temporal filtering effect.

However, increasing the bandwidth can result in oscillations, or electrical noise in the trace, which is the result of the inability of the circuit to distinguish between the current passing though the electrode into the cell and the current passing into the electrode capacitance into the bath. This type of noise can be reduced by lowering the bandwidth. This noise can also interfere in analysis of kinetics of certain types of events. For very small events, such as synaptic events, compensating has very little effect.

It is very important to monitor series resistance during your experiments, as it will change, that is quite normal. An initial series resistance should ideally be around 10% of the initial membrane resistance and/or about three times the open pipette resistance. This value should vary as little as possible throughout the experiment. It is commonly accepted that the series resistance should vary by less than 20% during electrophysiological recordings to be considered stable.

Why would the series resistance change?

Optimal long duration whole-cell recordings would see no changes in series resistance. However, the reality is that slight physical variation in the seal status (such as membrane resealing) can lead to changes in the access resistance. Therefore, changes in series resistance can be indicative of the seal quality or that the membrane is trying to close. This re-sealing affects the onset currents and therefore affects the peak of currents.

Should you compensate?

Several reports support the idea that series resistance compensation should be done for every recording. However, electrophysiologists seem to be divided as many experimenters balance the advantage of such compensation with the risk of introducing variability from cell to cell and increasing chances of losing cells due to unstable compensation and oscillation. Such discrepancy in methods remains a debate between electrophysiologists.

What is your experience of using series resistance compensations? What have you found in your own research? Tell us in the comments below and start a discussion on the topic. We are excited to hear your ideas and suggestions.